The evaluation of the spatial distributions of molecular marker levels in cells and tissues via immunohistological analyses constitutes a vital component of the diagnosis, prognosis and clinical management of human diseases including cancer. Nevertheless, immunohistological methods remain substantially restricted by the fact that only a few molecular markers can be examined on a single specimen. Considering that the size of clinical specimens can be small and that the number of informative molecular markers can be large, the types and number of molecular and cellular analyses that are actually performed on individual samples are frequently compromised. These issues limit current efforts to personalize the clinical management of cancer via molecular marker analyses. Furthermore, the present need to utilize multiple tissue sections or aspiration biopsies to examine multiple markers limits the ability to fully characterize individual rare cells and cellular niches. This project will surmount these problems by developing a new multiplexed and reiterative immunofluorescence imaging method called DNA-Catalyzed Molecular Biomarker Imaging and Amplification (DC-MBIA). Employing principles from the field of DNA-nanotechnology, DC-MBIA enables (1) the selective fluorophore labeling of multiple molecular probes (e.g., unique DNA-conjugated antibodies that direct nucleotide sequence-specific reactions of fluorophore-bearing DNA-complexes), (2) the stoichiometric amplification of fluorescent signals in the local proximity of a molecular marker, and (3) the removal of fluorophores from a sample via exceptionally-mild processing conditions. In this way, DC-MBIA permits fluorophore reutilization on a single specimen; the same types of fluorescent dye molecules can be selectively exchanged between molecular markers, and hence, distinct fluorescent channels of a microscope can now be used multiple times to image several sets of molecular markers, even if the markers of interest are present at low levels. While building the necessary infrastructure to facilitate this advance, this project will evaluate and optimize protocols for DC-MBIA to facilitate multiplexed and reiterative marker analyses. Here, the short-term feasibility goal is to demonstrate a minimum four-fold enhancement in the number of molecular markers that can be examined on a single specimen over that of current technologies (i.e., several tens of markers imaged, with line of sight to resolve hundreds). PUBLIC HEALTH RELEVANCE: The proposed project will create a new molecular probe technology that allows large numbers of molecular biomarkers to be examined on a single clinical biopsy, and hence, will improve the utility of biospecimens for the early detection and clinical management of cancer. Furthermore, the proposed technology will surmount current technological barriers that prohibit characterization of rare cells and low abundance markers within a single specimen, which in turn will lead to a better understanding of the molecular and cellular-level changes that occur within tumors, and assist in the future discovery of new targetable cancer markers.